靶向HER2的CAR-T细胞构建与抗肿瘤活性的体外分析  

Construction of HER2-specific CAR-T cells and in vitro analysis of their activity to suppress tumor cell growth

作　　者：李永强 姚崧源 李延胜 徐明恺[1] 张惠文[1] 张成刚[1] Yongqiang Li1,2, Songyuan Yao1,2, Yansheng Li1,2, Mingkai Xu1, Huiwen Zhang1, and Chenggang Zhang1(1 Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, Liaoning, China; 2 University of Chinese Academy of Sciences, Beijing 100049, Chin)

机构地区：[1]中国科学院沈阳应用生态研究所,辽宁沈阳110016 [2]中国科学院大学,北京100049

出　　处：《生物工程学报》2018年第5期731-742,共12页Chinese Journal of Biotechnology

基　　金：中国科学院战略性先导科技专项(A类)(No.XDA12020225);沈阳市科技计划项目(Nos.Z17-7-013;F14-150-9-00);国家科技重大专项重大新药创制项目(No.2012ZX09102301-013)资助~~

摘　　要：CAR-T细胞疗法通过靶向识别肿瘤细胞表面抗原,特异性杀伤肿瘤细胞,近年来已经成为肿瘤免疫疗法的研究热点。通过基因工程方法构建靶向人类表皮生长因子受体2(HER2)的CAR慢病毒表达质粒,以磷酸钙沉淀辅助多质粒共转染HEK293T细胞包装,制备CAR慢病毒颗粒lenti-car,感染人外周血单核细胞获得HER2靶向的CAR-T细胞,并分析其对HER2阳性和阴性肿瘤细胞的特异性抑制效果。研究结果表明,构建的CAR-T细胞可被HER-2阳性的肿瘤细胞特异性激活,分泌大量炎症性细胞因子IFN-γ和IL-2。在同样效靶比等处理条件下,构建的HER2靶向CAR-T细胞对HER2阳性的人卵巢癌细胞株SK-OV-3的生长抑制率为(58.47±1.72)%,显著高于对照组(P<0.05);而对HER2阴性的人慢性髓原白血病细胞株K562的生长抑制率为(11.74±2.37)%,与对照组无显著差异(P>0.05)。进一步,在K562细胞中转染人HER2表达载体使其成为HER2阳性,则HER2靶向CAR-T细胞对其的生长抑制率上升为(30.41±7.59)%,较HER2阴性K562具有明显差异(P<0.05)。研究结果表明,构建的HER2靶向的第二代CAR-T细胞可选择性地抑制高表达HER2蛋白的肿瘤细胞的生长,暗示了其对HER2阳性肿瘤进行细胞免疫治疗的临床应用前景。CAR-T cell therapy that targets surface antigens to kill tumor cells specifically has recently become another cornerstone in tumor immunotherapy. In this study, a lentiviral expression plasmid of CAR targeting human epidermal growth factor receptor 2（HER2） was constructed by genetic engineering. The recombinant plasmid was co-transfected with other packaging plasmids into HEK293 T cells by calcium phosphate precipitation to generate lenti-car, which are CAR lentiviral particles. HER2-specific CAR-T cells were obtained by transducing human peripheral blood mononuclear cells with lenti-car. Their specific inhibitory effects on HER2-positive and HER2-negative tumor cells were analyzed in vitro. The constructed CAR-T cells were specifically activated by HER2-expressing tumor cells as indicated by secretion of IFN-γ and IL-2. The inhibitory rate on HER2-positive SK-OV-3 cell line was（58.47±1.72）%, significantly higher than that on the mock-treated control group（P〈0.05）. The inhibitory rate on HER2-negative K562 cell lines was（11.74±2.37）%, which was not significantly different from that on the control group（P〉0.05）. Furthermore, when we transfected a HER2-expressing vector into K562, the inhibitory rate increased to（30.41±7.59）%, which was higher than that on HER2-negative K562（P〈0.05）. Thus, the constructed second-generation HER2-specific CAR-T cells specifically suppressed growth of tumor cells overexpressing HER2 protein, suggesting that HER2-specific CAR-T cells might prove useful for immunotherapy of HER2-positive cancer.

关 键 词：CAR-T细胞 人类表皮生长因子受体2(HER2) 肿瘤 免疫治疗 

分 类 号：R730.51[医药卫生—肿瘤]